**4. L(h)C biomass deconstruction to sugars**

L(h)C biomass, as said before, can be obtained at relatively low cost in different forms, representing a potential sugar source for the fermentative production of renewable fuels as well as other materials in modern biorefineries. Most of these potential applications rely on the predominant cellulose fraction susceptibility to enzymatic hydrolysis/acid hydrolyses or other structural changes.

However, the intrinsic nature of L(h)C materials is completely different from that found in starch. Starch granule serve as a temporary energy storage polymer with glycosidic linkages that can be readily hydrolyzed to supply glucose for germination and plant growth. Conversely, L(h)C has been projected by nature to function as a resistant structural material of carbohydrates and lignin that resist assault on cellulose through enzymatic deconstruction from a vast hydrolytic and/or oxidative enzymatic machinery secreted by various microorganisms, be fungal or bacterial. Besides to the protection conferred to cellulose by lignin and hemicellulose, factors such as cellulose crystallinity, low surface availability cellulolytic enzymes and degree of acetylation of hemicellulose prevent or hinder the whole polysaccharide deconstruction by microorganisms [25].

Dilute and hot aqueous acid pretreatment (e.g., H2

be discussed later in the chapter.

treated biomass [39, 40].

SO4 , H3 PO4

Sugar Versatility—Chemical and Bioprocessing of Many Phytobiomass Polysaccharides Using…

widely employed method on industrial scale [24]. Acid pretreatment is efficient in promoting hemicellulose removal where a rich C5 liquid fraction is generated, leaving a solid material with high content in cellulose and less lignin, being the residual cellulose fraction more stable in the acidic regime due to its crystalline structure. This pretreated material can be further submitted to enzymatic hydrolysis with an increased cellulase accessibility. Two general approaches in acid pT can be applied; high temperature (above 180°C) during less residence time (1–5 min) and lower temperature (<120°C) for long duration (30–90 min), respectively [24]. The most conventional commercially used acid is dilute sulfuric acid, mainly to its low cost. However, use of mainly strong acids such as sulfuric, hydrochloric and nitric acids, even diluted, presents various drawbacks. These are mainly related with an increased production of inhibitory compounds (furans like HydroxyMethylFurfuraldehyde (HMF) or its progressive degradation species such as levulinic and formic acids if more parameters severity is applied) and corrosion of reaction vessels [24, 25, 30]. Therefore, various other acids have been used to circumvent the harsh conditions imposed by strong acids, such as maleic acid [31], fumaric acid [31], oxalic acid [32] and, in our repetitive experiences, phosphoric acid, that will

In the alkaline pretreatments, L(h)C biomass is treated with alkali, normally sodium hydroxide or lime, Ca(OH)2, at normal temperature and pressure. The main advantage is the efficiency in lignin removal [24, 33, 34]. It also demands relatively low capital costs, allows lower inhibitors formation and ensures high glucose yields in the subsequent enzymatic hydrolysis step, besides the possibility of a better lignin recovery by co-addition of air or oxygen. In the

stream [33, 35]. Despite the inexpensiveness of lime and other hydroxides are inexpensive, downstream processing costs are high because the process requires a large quantity of water

Physico-chemical pretreatments include steam explosion pretreatment which is one of the most used methods for pretreatment of L(h)C biomass. In this approach, biomass is submitted to a high pressure (0.7–4.8 MPa) with saturated steam at high temperatures between 160 and 260°C for a few seconds followed by an explosive decompression, that cause separation of the fibers [24, 33, 36]. It can be carried out with or without (autohydrolysis) addition of an acid catalyst [37]. In autohydrolysis also named solvolysis, the high temperatures (160–250°C), an endogenous catalyst, acetic acid, is delivered from the O-Acetylated xylan and catalyzes the break of part of the xylo-backbone of hemicellulose thus producing of xylooligosacchrides along with some free D-xylose. The few side chain L-arabinofuranose substituents are completely released since their native *α*-configuration (allied to the furanosyl ring) are more prone to any acid hydrolysis [36]. Any alkaline pT can be performed with high total solids, providing higher yields, and a cellulose fraction with improved accessibility to enzymatic hydrolysis [38]. Some drawbacks of steam explosion pretreatment are the partial degradation of hemicelluloses, production of fermentation inhibitors (e.g. aromatic compounds, furfural and HMF) demanding a washing step for detoxification of the pre-

case of lime use, the mild catalyst may be recoverable injecting CO2

for the appropriate washing of the residual cellulose [24].

and organic acids) is the most

249

http://dx.doi.org/10.5772/intechopen.75229

in the liquefied alkaline

Therefore, more aggressive chemical and/or physical environments are required for the dismantlement of the L(h)C matrix, reducing the cellulose crystallinity, and increasing the porosity to a better accessibility to cellulose complex and more importantly, removing the hemicellulose and lignin barriers, which can be addressed to other technological goods. By doing this, the biomass become suitable for the production of second generation biofuels as ethanol, butanol, ABE (Acetone-butanol-ethanol) mix and other compounds like lactic acid, which can serve as a feedstock for the production of polylactic acid (PLA) to replace the petrochemical packaging materials such as polyethylene terephthalate (PET). In the case of cellulosic ethanol production four main steps must be taken in account: (1) biomass pretreatment; (2) enzymatic hydrolysis; (3) monosaccharide fermentation and (4) ethanol distillation.

There are several conditions for the selection of an appropriate L(h)C pretreatment (pT) method: (1) this should avoid the excessive size reduction of biomass particles, (2) preferably hemicellulose portion must be preserved as such (by alkaline pT), (3) converted do free xylose or xylo-oligosaccharides (acid pT) minimizing in this second case, the co-generation of degradation products like furfuraldehyde, a known inhibitor to microbial growth, (4) reduce the energy requirements, (5) explore a low-cost pT catalyst and/or inexpensive catalyst recycle and (6) preferably regenerating of any form of high-value lignin co-product [24].

Pretreatment can be performed by biological, physical and chemical processes and even combination of them. However, there are several possible combinations or operation modes. The choice of a particular method has to be based on a number of considerations, for example, which biomass will be used, the organism used for fermentation of the released sugars, and the costs implied. Common to all of these methods is that the L(h)C materials must be first mechanically pretreated (ground, chipped or milled) to increase the surface area.

In biological pretreatments, microorganism such as brown, white and soft rot fungi are used for degradation of lignin and hemicelluloses from biomass [26]. The efficiency of the process generally depends on the action of lignin-degrading enzymes such as peroxidases—lignin peroxidase (E.C. 1.11.1.7), manganese peroxidase (E.C. 1.11.1.7) and laccases (E.C. 1.10.3.2), generally copper-containing oxidases produced mainly by basidiomycetes. Supplementation with accessory enzymes like hemicellulases (endo/exoxylanases, *β*-D-xylosidases and *α*-Larabinofuranosidase) increase hydrolysis yields but also enzyme costs and dosages [27]. Although eco-friendly and without production of inhibitory compounds, biological pretreatment is not suitable for a pilot scale process mainly due energy demand and to the long incubation time for effective delignification [28]. However, biological pretreatment was found to be more effective if it is combined with another chemical or physical pretreatment [29].

Dilute and hot aqueous acid pretreatment (e.g., H2 SO4 , H3 PO4 and organic acids) is the most widely employed method on industrial scale [24]. Acid pretreatment is efficient in promoting hemicellulose removal where a rich C5 liquid fraction is generated, leaving a solid material with high content in cellulose and less lignin, being the residual cellulose fraction more stable in the acidic regime due to its crystalline structure. This pretreated material can be further submitted to enzymatic hydrolysis with an increased cellulase accessibility. Two general approaches in acid pT can be applied; high temperature (above 180°C) during less residence time (1–5 min) and lower temperature (<120°C) for long duration (30–90 min), respectively [24]. The most conventional commercially used acid is dilute sulfuric acid, mainly to its low cost. However, use of mainly strong acids such as sulfuric, hydrochloric and nitric acids, even diluted, presents various drawbacks. These are mainly related with an increased production of inhibitory compounds (furans like HydroxyMethylFurfuraldehyde (HMF) or its progressive degradation species such as levulinic and formic acids if more parameters severity is applied) and corrosion of reaction vessels [24, 25, 30]. Therefore, various other acids have been used to circumvent the harsh conditions imposed by strong acids, such as maleic acid [31], fumaric acid [31], oxalic acid [32] and, in our repetitive experiences, phosphoric acid, that will be discussed later in the chapter.

Conversely, L(h)C has been projected by nature to function as a resistant structural material of carbohydrates and lignin that resist assault on cellulose through enzymatic deconstruction from a vast hydrolytic and/or oxidative enzymatic machinery secreted by various microorganisms, be fungal or bacterial. Besides to the protection conferred to cellulose by lignin and hemicellulose, factors such as cellulose crystallinity, low surface availability cellulolytic enzymes and degree of acetylation of hemicellulose prevent or hinder the whole polysaccha-

Therefore, more aggressive chemical and/or physical environments are required for the dismantlement of the L(h)C matrix, reducing the cellulose crystallinity, and increasing the porosity to a better accessibility to cellulose complex and more importantly, removing the hemicellulose and lignin barriers, which can be addressed to other technological goods. By doing this, the biomass become suitable for the production of second generation biofuels as ethanol, butanol, ABE (Acetone-butanol-ethanol) mix and other compounds like lactic acid, which can serve as a feedstock for the production of polylactic acid (PLA) to replace the petrochemical packaging materials such as polyethylene terephthalate (PET). In the case of cellulosic ethanol production four main steps must be taken in account: (1) biomass pretreatment; (2) enzymatic hydrolysis;

There are several conditions for the selection of an appropriate L(h)C pretreatment (pT) method: (1) this should avoid the excessive size reduction of biomass particles, (2) preferably hemicellulose portion must be preserved as such (by alkaline pT), (3) converted do free xylose or xylo-oligosaccharides (acid pT) minimizing in this second case, the co-generation of degradation products like furfuraldehyde, a known inhibitor to microbial growth, (4) reduce the energy requirements, (5) explore a low-cost pT catalyst and/or inexpensive catalyst recycle

Pretreatment can be performed by biological, physical and chemical processes and even combination of them. However, there are several possible combinations or operation modes. The choice of a particular method has to be based on a number of considerations, for example, which biomass will be used, the organism used for fermentation of the released sugars, and the costs implied. Common to all of these methods is that the L(h)C materials must be first

In biological pretreatments, microorganism such as brown, white and soft rot fungi are used for degradation of lignin and hemicelluloses from biomass [26]. The efficiency of the process generally depends on the action of lignin-degrading enzymes such as peroxidases—lignin peroxidase (E.C. 1.11.1.7), manganese peroxidase (E.C. 1.11.1.7) and laccases (E.C. 1.10.3.2), generally copper-containing oxidases produced mainly by basidiomycetes. Supplementation with accessory enzymes like hemicellulases (endo/exoxylanases, *β*-D-xylosidases and *α*-Larabinofuranosidase) increase hydrolysis yields but also enzyme costs and dosages [27]. Although eco-friendly and without production of inhibitory compounds, biological pretreatment is not suitable for a pilot scale process mainly due energy demand and to the long incubation time for effective delignification [28]. However, biological pretreatment was found to be more effective if it is combined with another chemical or physical pretreatment [29].

and (6) preferably regenerating of any form of high-value lignin co-product [24].

mechanically pretreated (ground, chipped or milled) to increase the surface area.

ride deconstruction by microorganisms [25].

248 Sugarcane - Technology and Research

(3) monosaccharide fermentation and (4) ethanol distillation.

In the alkaline pretreatments, L(h)C biomass is treated with alkali, normally sodium hydroxide or lime, Ca(OH)2, at normal temperature and pressure. The main advantage is the efficiency in lignin removal [24, 33, 34]. It also demands relatively low capital costs, allows lower inhibitors formation and ensures high glucose yields in the subsequent enzymatic hydrolysis step, besides the possibility of a better lignin recovery by co-addition of air or oxygen. In the case of lime use, the mild catalyst may be recoverable injecting CO2 in the liquefied alkaline stream [33, 35]. Despite the inexpensiveness of lime and other hydroxides are inexpensive, downstream processing costs are high because the process requires a large quantity of water for the appropriate washing of the residual cellulose [24].

Physico-chemical pretreatments include steam explosion pretreatment which is one of the most used methods for pretreatment of L(h)C biomass. In this approach, biomass is submitted to a high pressure (0.7–4.8 MPa) with saturated steam at high temperatures between 160 and 260°C for a few seconds followed by an explosive decompression, that cause separation of the fibers [24, 33, 36]. It can be carried out with or without (autohydrolysis) addition of an acid catalyst [37]. In autohydrolysis also named solvolysis, the high temperatures (160–250°C), an endogenous catalyst, acetic acid, is delivered from the O-Acetylated xylan and catalyzes the break of part of the xylo-backbone of hemicellulose thus producing of xylooligosacchrides along with some free D-xylose. The few side chain L-arabinofuranose substituents are completely released since their native *α*-configuration (allied to the furanosyl ring) are more prone to any acid hydrolysis [36]. Any alkaline pT can be performed with high total solids, providing higher yields, and a cellulose fraction with improved accessibility to enzymatic hydrolysis [38]. Some drawbacks of steam explosion pretreatment are the partial degradation of hemicelluloses, production of fermentation inhibitors (e.g. aromatic compounds, furfural and HMF) demanding a washing step for detoxification of the pretreated biomass [39, 40].

Most of the demonstration plants installed worldwide aiming cellulosic ethanol production is based on steam explosion pretreatment, or its variations. One example is POET-DSM Advanced Biofuels that constructed a cellulosic biorefinery alongside the POET Biorefining - Emmetsburg plant. The company contracted ANDRITZ Inc. to supply a two-step biomass treatment process that includes a vertical reactor and a continuous steam explosion (SE) technology to pretreat corn residues (stalks, husks, leaves and cob). In Brazil, GranBio began operations in 2014 with 22 million gallons per year cellulosic ethanol facility, Bioflex 1, using Beta Renewables' PROESA pretreatment process, Novozymes' cellulase enzymes, and DSM's yeasts. In PROESA technology plant, the biomass is pretreated with steam (high temperature and pressure) without chemical addition followed by enzymatic hydrolysis (viscosity reduction and hydrolysis). Some companies have reported difficulties regarding biomass feeding/transport and high degree of equipment wear due to the frictional effect of abrasive materials present in biomass [41]; signaling that this kind of pretreatment still remains as a challenge.

enough to produce the precious FOS—FructoOligoSacchrides), xylan, mannan and microalgal glucans are pretreated under more severe parameters (thermopressurized reactor) in the range of 2 atm (120°C) to 15.5 (200°C) for a short peaking time (1–2 min) but always keeping the effective oPA concentration with a narrow pH 3.0–1.5 range and more often, at pH 2 or its

Almost four decades ago, initial studies with L(h)C biomass (rye grass straw) treated with phosphoric acid was carried out to verify the amount of sugar and yeast fermentability after

SO4

acceptability by rodents [47]. They have shown that both fermentability (after neutralization with ammonia) and rodent palatability were highest when the straw was treated with

PO4

toxic degradation products of monomeric sugars, such as furfural and HMF.

PO4

PO4

end of 1970s and was further acquired by Novo Nordisk's in 2003.

is superior to H2

sugar per g of straw. They also verified that if straw were treated with higher concentrations

Prof J.D. Fontana's group at LQBB—Biomasses Chemo/Biotechnological Laboratory formerly at UFPR (Federal University of Paraná) and now at UTFPR (Federal Technological University of Parana, Curitiba-PR, Brazil) has been consolidating for a long time the phosphoric pretreat-

tion. An initial study focused on the pretreatment of sugarcane and sorghum bagasses with

or partial hydrolysis of hemicellulose fraction to xylose>xylo-oligosaccharides>arabinose depending on the variation of the severity parameters just before mentioned. Moreover, it was observed improved fermentation of the solubilized pentoses to ethanol and acetic acid by *Pachysolen tannophilus* and to ethanol by *Fusarium oxysporum*. *Pachysolen tannophilus* was the first yeast shown to be capable to convert xylose directly to ethanol under anaerobic conditions (with the concomitant production of xylitol and acetic acid) [49], while *F. oxysporum* is one of the few fungal species reported to ferment plant carbohydrate polymers to ethanol in just one-step process [50]. It was observed that fermentation capability was related to lignin solubilization followed by its removal using ethyl acetate or activated charcoal. Most importantly, phosphoric acid pretreatment on sugarcane and sorghum bagasses allowed almost complete conversion of cellulose to glucose using commercial cellulases produced at that time by Biobras (BIOFERM, Brazil), a Brazilian company that operated a 2G ethanol plant in the

Interestingly, even past >30 years, our oPA technology for sugarcane processing has been used with industrial proposals, as, for example, in the CANEBIOFUEL (Conversion of sugar cane biomass into ethanol) Project, that was funded by the European Commission (FP7-Energy) which planned to obtain a deeper knowledge and a scientific and technological platform for converting sugarcane biomass into fermentable sugars. The project concluded that, in general, lower severity during pretreatment, with lower temperatures and shorter times, result in better glucose yield than the opposite. Primarily based on ease of enzyme hydrolysis it was

SO4

for the production of bioethanol [48]. The main results from this pretreatment using

, HCl and H3

Sugar Versatility—Chemical and Bioprocessing of Many Phytobiomass Polysaccharides Using…

or HCl, yeast yield declined probably due to the higher concentration of

PO4

(30 min at 121°C), which produced 0.25 g of

, alone and under moderated thermopressuriza-

and 200°C, 3 atm, during 2 min) was a complete

for the acid catalyzed pretreatment. However,

aiming to study the feed

http://dx.doi.org/10.5772/intechopen.75229

251

pKa value (2.1).

(>0.5 N) of H2

also found that H3

PO4

H3 PO4

treatments with various concentrations of H2

a combination of 0.23 N HCl and 0.15 N H3

ment technology using very diluted H3

optimized conditions (0.065% v/v H3

SO4

Other important pretreatments comprise ammonia freeze explosion (AFEX) [42], liquid hot water [43], organosolv and ionic liquids [44], the last one implying in higher costs despite the alternative of catalyst recycling. Also, new pretreatment technologies are constantly being developed, such as sub-/supercritical water [45] and supercritical carbon dioxide [46].
